Genotypic Diversity and Epidemiology of Human Rhinovirus Among Children With Severe Acute Respiratory Tract Infection in Shanghai, 2013-2015.

Human rhinovirus (HRV), and particularly HRV-C, is increasingly recognized as a cause of severe acute respiratory infections (SARIs). However, little is known about the genotypic diversity and epidemiology of HRV among children with SARI. Thus, we investigated the genotypic diversity and epidemiology of HRV in children with SARI in China over a 2-year period. In total 1,003, nasopharyngeal aspirates were collected from children hospitalized with SARI in Shanghai from 2013 to 2015. HRV was screened for by a PCR method targeting the viral 5′ UTR and was genotyped by sequencing of the VP4–VP2 region of the HRV genome. We also screened for 15 other common respiratory viruses to assess the prevalence of co-infection with HRV. The patient demographic and clinical data were reviewed. HRV was detected in 280 (27.9%) of the 1,003 specimens: HRV-A in 140 (14.0%), HRV-B in 21 (2.1%), HRV-C in 56 (5.6%), and HRV-untyped in 63 (6.3%). A phylogenetic analysis identified 77 genotypes (43 HRV-A, 10 HRV-B, and 24 HRV-C), among which A78, A12, A89, B70, C2, C6, and C24 predominated. HRV-A was detected mainly in winter 2013 and autumn 2014, while HRV-C detection peaked in autumn 2013 and 2014. The detection frequency of HRV-A was highest in patients <5 years old. Most HRV co-infections involved adenovirus, human bocavirus, and/or human respiratory syncytial virus. In conclusion, HRV-A and -C predominate in children with SARI in Shanghai. Among the 77 genotypes detected, A78, A12, A89, B70, C2, C6, and C24 were the most frequent. The HRV species responsible for SARIs differs according to season and age.


INTRODUCTION
Human rhinovirus (HRV), a single-stranded positive-sense RNA virus, belongs to the genus Enterovirus and family Picornaviridae, and is classified as HRV-A, -B, or -C. HRV-A and HRV-B were discovered in the 1950s (Price, 1956), while HRV-C was identified using molecular techniques in 2006 (Lamson et al., 2006;Lau et al., 2007). According to the 2017 International Committee on the Taxonomy of Viruses (ICTV) release 1 , a total of 168 HRV genotypes (80 HRV-A, 32 HRV-B, and 56 HRV-C) are recognized (Kuroda et al., 2015).
Human rhinovirus genomic RNA is approximately 7.2 kb that consisting of a single open reading frame (ORF) encodes 11 proteins, with 5 and 3 untranslated regions (UTR) at both end, respectively. The ORF encodes a poly-protein which is cleaved by viral proteases to produce 11 proteins including four structural viral proteins (VP) 1 to 4. Compared to the rest of the HRV genome, the capsid proteins exhibit a high degree of heterogeneity resulting in a wide range of antigenic diversity. RT-PCR assays targeted the 5 -UTR are usually used for HRV clinical detection. HRV species and types are classified almost exclusively now based on VP1 or VP4/VP2 sequence alignments (Wisdom et al., 2009).
Human rhinovirus is a frequently detected respiratory virus in children with mild acute respiratory infection (ARI), but may also lead to more-severe respiratory tract symptoms, such as pneumonia, bronchiolitis, and asthma. HRV is, after respiratory syncytial virus (RSV), the second most frequent viral cause of community-acquired pneumonia and other severe acute respiratory infections (SARIs) (Honkinen et al., 2012;Esposito et al., 2013). HRV-C is more frequently associated with wheezing episodes, asthma exacerbations, and lower respiratory tract infections compared with HRV-A and -B (Linsuwanon et al., 2009;Gern, 2010;Bizzintino et al., 2011). However, there is reportedly no relationship between disease severity and HRV species (Lee et al., 2012;Chen et al., 2015;van der Linden et al., 2016).
Data on the genotypic diversity and epidemiology of HRVs in children with SARI are sparse. Thus, we evaluated the predominant HRV species and genotypes, and their associations with the clinical characteristics, of 1,003 children hospitalized with SARI from 2013 to 2015 in Shanghai, China.

Ethics Issues
All aspects of the study were performed in accordance with the national ethics regulations and approved by the Ethics Committee of the Children's Hospital of Fudan University (Jun Shen; CHFU2013016) as well as the Ethics Committee of National Institute for Viral Disease Control and Prevention (RL; IVDC2013022). Participants were received "Written Informed Consent" on the study's purpose and of their right to keep information confidential. Written consent was obtained from all participants or their guardians.

Patients and Sample Collection
From June 2013 to August 2015, 1,003 nasopharyngeal aspirates (NPAs) were collected from children hospitalized with SARI in the Children's Hospital of Fudan University, Shanghai, China. The revised World Health Organization SARI case definition (World Health Organization [WHO], 2011) was 1 http://ictvonline.org/virusTaxonomy.asp adopted and cases with clinical suspicion of SARI for children was enrolled (Zhang et al., 2013;Wang et al., 2016). Eligibility and classification of the clinical syndromes of SARI were determined from individual's original record of medical history and examination. The criteria of hospitalized patient inclusion were: sudden onset of fever >38 • C and cough or sore throat and difficulty breathing (dyspnea, oxygen saturation < 90%). Additional criteria were a normal or low leukocyte count, or lower chest wall indrawing. Demographic data and clinical findings at the time of diagnosis were recorded on a standard form. All NPA samples were stored at −80 • C until use.

HRV Detection and Genotyping
Viral nucleic acid was extracted from 200 µL of sample using QIAamp MinElute Virus Spin Kits (Qiagen, Germany). cDNA was synthesized using an AMV reverse transcriptase and random hexamer primers (Promega, United States), as described previously (Lu et al., 2012). Nested RT-PCR targeting of the 5 -UTR was employed for HRV screening, and of the VP4-VP2 regions for genotyping. All (totally nine, except HEV IS only) of HRV or HRV/HEV primers from 5-UTR to VP4-VP2 were used to detect HRV, as described previously (Wisdom et al., 2009). Specimens from which amplification of the VP4-VP2 regions failed were defined as untyped. PCR products were confirmed by sequencing. Phylogenetic analysis was conducted using Molecular Evolutionary Genetics Analysis (MEGA) software (ver. 7).

Detection of Common Respiratory Viruses
Human rhinovirus-positive specimens were screened for influenza virus types A and B, parainfluenza virus types 1 to 3, RSV, picornaviruses (enteroviruses and rhinoviruses), and adenovirus (AdV) using three multiple-nested-PCR assays; and for human metapneumovirus (hMPV), human bocavirus (HBoV), and human coronavirus 229E/OC43/NL63/HKU1 using nested-PCR assays. The multiple-nested and nested PCRs were performed as described previously (da Silva Filho et al., 2012;Lu et al., 2012). All PCR products of 15 common respiratory viruses were confirmed by sequencing.

Statistical Analysis
Data analysis was performed using PASW (ver. 18; SPSS Inc., United States) and GraphPad Prism software (ver. 5.0; GraphPad Software, Inc., United States). Age, maximum body temperature, laboratory parameters, clinical features, and HRV prevalence were compared by χ 2 -test or Fisher's exact test for categorical variables, and by two-tailed paired Student's t-test for continuous variables. A value of P < 0.05 was considered indicative of statistical significance.

Epidemiology of HRV
In total, 1,003 NPAs were collected. The male: female ratio was 609:394 (1.55:1) and the median age was 1 year (range: 10 days to 15 years). HRV was detected in 280 (27.9%) of the 1,003 specimens: HRV-A in 140 (14.0%), HRV-B in 21 (2.1%), HRV-C in 56 (5.6%), and HRV-untyped in 63 (6.3%). The HRV detection rates are shown in Table 1. The detection rates of HRV-A and -B differed significantly (P < 0.001). In general, the 5 -UTR region highly conserved between HRV and enterovirus, causing cross-reactivity in RT-PCR assay for the two viruses. In the current study, 42 enterovirus positive samples also were detected by nested RT-PCR targeting of the HRV 5 -UTR.
Human rhinovirus infected significantly more males than females (65.7% vs. 34.3%, P = 0.044), and HRV was detected in patients of all ages, although HRV-B was not detected in those aged 3-4 years ( Table 1). The detection frequency of HRV-A was significantly lower in patients aged 5-9 years (P = 0.008), but the frequencies of detection of HRV-B, -C, and -untyped did not differ according to age.
The seasonal distribution of HRV is shown in Figure 1.

Clinical Characteristics
The clinical characteristics of the patients with HRV infections are listed in Table 2. All HRV-positive patients presented with SARI. Fever, cough, gasping, cyanosis, and diarrhea were the most common symptoms at presentation. There was no significant difference in clinical symptoms according to HRV species, except that cough and cyanosis were less frequent in patients infected with HRV-A and HRV-C, respectively (P = 0.048 and 0.005).

Co-infection With Other Respiratory Viruses
Of the 280 HRV-positive patients, 131 (46.8%) were co-infected-102 with one other virus, 21 with two other viruses, and 8 with three other viruses ( Table 3). Most such co-infections involved AdV, HBoV, and RSV (39.7, 38.2, and 17.6%, respectively). The patients with co-infections did not have more serious disease (data not shown).
Human rhinovirus circulated throughout this 2-year study with peaks in winter 2013 and autumn 2014; HRV-C predominated in autumn. This seasonal pattern is similar to those reported previously (Xiang et al., 2010;Marcone et al., 2014;van der Linden et al., 2016). Also, HRV-A was detected most frequently in those <5 years old, while the frequency of detection of HRV-B and -C did not differ by age. In contrast, in Mongolia, HRV-A and -C are detected more frequently in younger children, and -B more frequently in older children (Tsatsral et al., 2015).
Human rhinovirus-A and HRV-C are reportedly associated with more severe illness (Miller et al., 2011;Fawkner-Corbett et al., 2016); however, others have reported no link between species and disease severity (Xiang et al., 2010;Henquell et al., 2012;Rahamat-Langendoen et al., 2013;Marcone et al., 2014). In this study, there were no significant differences in clinical symptoms among the three HRV species, except that cough and cyanosis were less frequent in patients with HRV-A and HRV-C, respectively. HRV-C infection in boys under 5 years old with acute asthma significantly increases the risk of moderate/severe exacerbations (Lambert et al., 2017). Moreover, of 570 HRV-positive hospitalized patients with community-acquired pneumonia, 57 (10%) had viremia; the vast majority (98.2%) of viremic patients were infected with HRV-C. The frequency of HRV viremia was higher in patients 1-2 years of age, and patients with viremia were more likely to have severe clinical symptoms, such as chest retraction, wheezing, and asthma (Lu et al., 2017). Therefore, particular HRV species may be associated with disease severity at specific situations which further research is warranted.
Almost half (46.8%) of the HRV-positive patients were coinfected with other respiratory viruses, most frequently ADV, HBoV, and hRSV. This may reflect the viruses in circulation at the time, consistent with previous reports (Xiang et al., 2010;van der Linden et al., 2016;Blaschke et al., 2018). Additionally, for those patients infected with 2-4 respiratory viruses, further research is necessary to demonstrate the dominant virus by qPCR positive with CT value and impact on disease severity.
In summary, we report for in detail the first time the variety of HRV genotypes in circulation and their distribution according to season and age group, as well as their clinical symptoms, in children hospitalized with SARI in China during a 2-year period. Our data shown that HRV was detected in 280 (27.9%) of the 1,003 NPAs from children with SARI in Shanghai, in which HRV-A and -C were predominate. Among the 77 HRV genotypes detected, A78, A12, A89, B70, C2, C6, and C24 were the most frequent. The HRV species responsible for SARIs differs according to season and age.